The Research will entail an extensive series of studies on the 5' flanking regions of the light (NF_L) mid-sized (NF-M) and heavy (NF-H) neurofilament genes in order to identify the cis-acting elements and trans-acting factors that regulate the cell-specific expression of neurofilament genes. Promoter/enhancer elements of the NF genes will be fully sequenced. Defined DNA fragments, including deletion mutants, will be inserted into chimeric plasmids bearing the chloramphenicol transferase (CAT gene and the chimeric plasmids will be used to test the function of NF promoter/enhancer elements during transient transfections of neuronal and non-neuronal cell lines. Nuclear extracts from the same cell lines will be studied in footprinting and gel shift assays in order to map the common and unique binding sites along the 5' flanking regions of the NF genes. Correlation of binding sites with functional assays of the same elements in neuronal vs non-neuronal cells will be undertaken to identify the cis-acting elements and trans-acting factors that lead to specific expression of each NF gene.